Lipase-producing Strains Research Articles

Novel extracellular lipase gene Lip1728 influences nutrient-dependent performance bacterial quorum sensing of Burkholderia pyrrocinia WZ10-3

Quorum sensing (QS) is a cellular communication mechanism in which bacteria secrete and recognize signaling molecules to regulate group behavior. Lipases provide energy for bacterial cell growth but it is unknown whether they influence nutrient-dependent QS by hydrolyzing substrate. A high-yield lipase-producing strain, Burkholderia pyrrocinia WZ10–3, was previously identified in our laboratory, but the composition of its crude enzymes was not elucidated. Here, we identified a key extracellular lipase, Lip1728, in WZ10–3, which accounts for 99 % of the extracellular lipase activity. Lip1728 prefers to hydrolyze triglycerides at sn-1,3 positions, with pNP-C16 being its optimal substrate. Lip1728 exhibited activity at pH 5.0–10.0 and regardless of the presence of metal ions. It had strong resistance to sodium dodecyl sulfate and short-chain alcohols and was activated by phenylmethanesulfonylfluoride (PMSF). Lip1728 knockout significantly affected lipid metabolism and biofilm formation in the presence of olive oil. Finally, oleic acid, a hydrolysate of Lip1728, influenced the production of the signal molecule N-acyl homoserine lactone (AHL) and biofilm formation by downregulating the AHL synthetase gene pyrI. In conclusion, Lip1728, as a key extracellular lipase in B. pyrrocinia WZ10–3, exhibits superior properties that make it suitable for biodiesel production and plays a crucial role in QS.

Preparation, purification, and biochemical of fat-degrading bacterial enzymes from pig carcass compost and its application

BackgroundA lot of kitchen waste oil is produced every day worldwide, leading to serious environmental pollution. As one of the environmental protection methods, microorganisms are widely used treating of various wastes. Lipase, as one of the cleaning agents can effectively degrade kitchen waste oil. The composting process of pig carcasses produces many lipase producing microorganisms, rendering compost products an excellent source for isolating lipase producing microorganisms. To our knowledge, there are no reports isolating of lipase producing strains from the high temperature phase of pig carcass compost.MethodologyLipase producing strains were isolated using a triglyceride medium and identified by 16S rRNA gene sequencing. The optimal fermentation conditions for maximum lipase yield were gradually optimized by single-factor tests. The extracellular lipase was purified by ammonium sulfate precipitation and Sephadex G-75 gel isolation chromatography. Amino acid sequence analysis, structure prediction, and molecular docking of the purified protein were performed. The pure lipase's enzymatic properties and application potential were evaluated by characterizing its biochemical properties.ResultsIn this study, a lipase producing strain of Bacillus sp. ZF2 was isolated from pig carcass compost products, the optimal fermentation conditions of lipase: sucrose 3 g/L, ammonium sulfate 7 g/L, Mn2+ 1.0 mmol/L, initial pH 6, inoculum 5%, temperature 25 ℃, and fermentation time 48 h. After purification, the specific activity of the purified lipase reached 317.59 U/mg, a 9.78-fold improvement. Lipase had the highest similarity to the GH family 46 chitosanase and molecular docking showed that lipase binds to fat via two hydrogen bonds at Gln146 (A) and Glu203 (A). Under different conditions (temperature, metal ions, organic solvents, and surfactants), lipase can maintain enzymatic activity. Under different types of kitchen oils, lipase has low activity only for ‘chicken oil’, in treating other substrates, the enzyme activity can exceed 50%.ConclusionsThis study reveals the potential of lipase for waste oil removal, and future research will be devoted to the application of lipase.

Production, optimization, and physicochemical characterization of biodiesel from seed oil of indigenously grown Jatropha curcas

With the growing demand for vegetable oils, alternative non-edible feedstocks like Jatropha curcas seed oil have gained interest for biodiesel production. The study aimed to comprehensively evaluate the physicochemical properties and biodiesel production potential of locally produced J. curcas seeds in Pakistan. Two different approaches were applied: a chemical synthesis approach involving acidic pretreatment and alkaline transesterification, and a biosynthetic approach using a lipase-producing strain of the Bacillus subtilis Q5 strain. The microbial biosynthesized biodiesel was further optimized using the Plackett–Burman design. The physicochemical properties of the J. curcas methyl esters were analyzed to assess their suitability as biodiesel fuel. Initially, the raw oil had a high free fatty acid content of 13.11%, which was significantly reduced to 1.2% using sulfuric acid pretreatment, keeping the oil to methanol molar ratio to be 1:12. Afterward, alkaline transesterification of purified acid-pretreated seed oil resulted in 96% biodiesel yield at an oil to methanol molar ratio of 1:6, agitation of 600 revolutions per minute (RPM), temperature 60°C, and time 2 h. Moreover, alkaline transesterification yielded ∼98% biodiesel at the following optimized conditions: oil to methanol molar ratio 1:6, KOH 1%, time 90 min, and temperature 60°C. Similarly, the Bacillus subtilis Q5 strain yielded ∼98% biodiesel at the following optimized conditions: oil: methanol ratio of 1:9, agitation 150 RPM, inoculum size 10%, temperature 37°C, and n-hexane 10%. The fuel properties of J. curcas seed biodiesel are closely related to standard values specified by the American Society for Testing and Materials (ASTM D6751–20a), indicating its potential as a viable biodiesel fuel source.

Screening for Extremophilic Lipase Producing Bacteria: Partial Purification and Characterization of Thermo-Halophilic, Solvent Tolerant Lipase from Bacillus sp.

The introduced study aims to isolate potent lipase producing strain from extreme habitats. The aim is extended to characterize the produced enzyme with respect to the effect of salt, temperature, pH and different organic solvents. Tween 80 plate method was used for discriminating lipase producing strains in the isolation step. Lipase activity of the selected isolates was confirmed through Rhodamine B plate assay. The enzyme of the most potent isolate was partially purified using 80% ammonium sulphate, quantified, and characterized using the synthetic substrate, para nitrophenyl laurate (pNP-C12). Twenty-three lipase-producing isolates were screened from different soil samples in Wadi El-Natrun, Egypt. Among the tested strains, a thermo- halo-tolerant bacterial isolate identified as Bacillus sp. was the most potent in terms of lipase production under high temperature and high salt concentration. The enzyme displayed its optimal activity at 60 oC and 2 M NaCl. Moreover, it showed a robust stability in 4M NaCl up to 2 h incubation time. Non-polar solvents have significantly raised the enzyme activity by 300% and 350% for ethyl acetate and n-hexane, respectively. The obtained data introduces useful insights for applying lipases in chemical catalysis and food processing fields.

The potential of biodiesel production from grasses in Thailand through consolidated bioprocessing using a cellulolytic oleaginous yeast, Cyberlindnera rhodanensis CU-CV7

Biodiesel can be produced from lipids derived from the cellulolytic oleaginous yeast Cyberlindnera rhodanensis CU-CV7 when directly cultivated on undetoxified Thai-grass hydrolysates through consolidated bioprocessing (CBP). The lipid content of CU-CV7 grown on a glucose-based medium was 26.77 ± 0.56% of the cell dry weight (CDW). Strain CU-CV7 is a great CBP-enabling microbe because of its native capacity to generate lipid (22.19 ± 0.72% of CDW) while simultaneously expressing endoglucanase, exoglucanase, and β-glucosidase activities (0.33, 0.06, and 0.20 IU/mL, respectively) on carboxymethylcellulose. Strain CU-CV7 was demonstrated to be a lipase-producing strain and could potentially be employed as a biocatalyst to produce biodiesel with a maximum extracellular lipase activity of 1.47 IU/mL. The lipid production from 13 types of grass without the need for additional cellulase was evaluated. The highest lipid yield was obtained from Napier (Lampang ecotype; 1.01 ± 0.14 g/L) and was comprised of a high proportion of saturated fatty acids. The predicted biodiesel properties were found to be suitable with respect to the biodiesel international standards. Thus, lipid production from these grasses by CU-CV7 appears to open a new path for the renewable biodiesel industry and to satisfy the SDG 7 and SDG 13 goals.

Enterococcus spp. in gut microbiota of patients with pulmonary tuberculosis

Aim. To defne the role of Enterococcus spp. in the gut microbiota ensemble in patients with pulmonary tuberculosis. Materials andMethods. Inoculation of intestinal cultures was performed in 64 patients with multidrug-resistant tuberculosis before the specific therapy (30 patients and 55 strains) and after 1 month of the treatment (34 patients and 63 strains). We then assessed the production of haemolysins, gelatinase, phospholipase, adhesion factors, and organic acids.Results. A high prevalence of intestinal dysbiosis (66.7%) were detected in patients before the start of anti-tuberculosis therapy. Specifc treatment was associated with a decrease in Lactobacillus spp. along with an increase in C. perfringens and Candida spp. titers as well as with higher prevalence of E. coli lac. Before the treatment, Enterococcus spp. showed pronounced adhesion properties, providing colonization of the intestinal mucosa in titers of 6 (4; 7) lg CFU/g, although not producing invasion enzymes. Upon the 1 month of anti-tuberculosis treatment, adhesion capability reduced and 36% of the strains were low-adhesive. An increase of lipase-producing strains to 18% suggested the adaptation of Enterococcus spp. to a high content of lipids and fatty acids in the intestine.Conclusion. In patients with pulmonary tuberculosis having high prevalence of intestinal dysbiosis, Enterococcus spp. are frequent symbionts well integrated into the gut microbiota.

Digging and identification of novel microorganisms from the soil environments with high methanol-tolerant lipase production for biodiesel preparation

Converting renewable biomass into carbon-neutral biofuels is one of the most effective strategies to achieve zero carbon emissions and contribute to environmental protection. Microorganisms from the soil were primarily screened on the rhodamine B-plate for highly-active lipase producing strains and re-screened on a tributyrin-methanol plate using crude lipases produced from the initially screened-out strains. The lipase-producing strains with higher methanol-tolerant lipase were identified based on morphological characteristics and 16S rDNA sequencing. The crude lipases with much higher methanol-tolerance from screened top-4 strains, Stenotrophomonas maltophilia D18, Lysinibacillus fusiformis B23, Acinetobacter junii C69, and A. pittii C95 showed temperature optima of 25 °C, 35 °C, 30 °C, and 30 °C at pH 7.0, respectively, while their pH optima were 8.0, 7.0, 7.5, and 7.5 at each optimum temperature, respectively. After 24-h incubation, they retained more than 85% of their original activities in 25%, 15%, 20%, and 20% of methanol, respectively. They catalyzed the conversion of soybean oil into biodiesel by yields of 63.1%, 35.4%, 74.6%, and 78.5% after 24-h reactions, respectively. In conclusion, the as-isolated microorganisms producing high methanol-tolerant lipase are considered promising to provide robust biocatalyst for efficient biodiesel preparation and other industrial applications.

Characterization of a lipolytic strain of Pseudomonas stutzeri SN-3 and production of triacylglycerol hydrolase with concomitant biodegradation of palm oil

Lipases are triacylglycerol hydrolases (3.1.1.1) that under aqueous condition catalyzes the hydrolysis of triglycerides. Lipases are the ubiquitous enzymes with their applications ranging from food industry to cosmetics, pharmaceuticals and bioremediation purposes. The present research involves 16S rDNA sequencing of a lipase producing strain isolated indigenously. The strain was identified as a novel Pseudomonas stutzeri SN-3, the gene sequence of which was deposited in GenBank with accession number MH639065. The research design also includes the exploration of alternative fermentation conditions for maximum production of triacylglycerol hydrolase from the novel strain. Different physical and chemical parameters were studied which includes temperature, pH, fermentation time course, nitrogen sources, carbon sources, phosphate sources, different salts and their concentrations for getting optimal yield of triacylglycerol hydrolase. Utilization of olive oil for production of lipases is a conventional approach, which is quite costly for commercial applications; therefore, palm oil was incorporated in cultivation medium as an alternative cheap substrate for triacylglycerol hydrolase production. Enzyme yield from Pseudomonas stutzeri SN-3 was optimized by using 1 gm% palm oil and 4 gm% yeast extract as carbon and nitrogen sources respectively in the presence of 2 gm% CaCl2 as enzyme stabilizer and 0.01% KH2PO4 as bacterial growth promoter. The maximum enzyme production was observed after 48 hours of fermentation with medium pH 7 at 37 ⁰C. Conclusively, we had a novel Pseudomonas stutzeri SN-3 specie and a cost-effective and eco-friendly medium for commercial production of triacylglycerol hydrolase.

Process parameter studies by central composite design of response surface methodology for lipase activity of newly obtained Actinomycete

Actinomycete strains from cottonseed soap-stock (a dark gelatinous waste from oil refinery) were obtained. Cultures were analyzed for the enzyme lipase, protease and cellulase production using plate assay(s). Best lipolytic isolates were further quantitatively analyzed for lipase production, using substrate p-nitrophenyl palmitate (p-NPP). Cultures were characterized by morphological and molecular studies. The potent lipase producing strain of actinomycete was optimized for pH, temperature, inoculum size, carbon and nitrogen sources. Further, the lipolytic strain of actinomycete was grown on synthetic media and growth factors were optimized by RSM (Response Surface Methodology) for maximum production of lipase. Total 49 cultures were obtained from enriched soap-stock samples; among them 7 isolates were strains of actinomycete. Potent actinomycete strains were identified as Nocardiopsis alba , Streptomyces leeuwenhoekii , Streptomyces caelestis , Streptomyces werraensis and Streptomyces sclerotialus . Based on quantitative lipase production study, Nocardiopsis alba was selected as the best lipase producer. Optimized results of process parameter studies for lipase production by Nocardiopsis alba were: 5mL inoculum size, temperature 40 ∘ C, shaking at 130 rpm, pH 8.0 and cottonseed oil as the best carbon source. Cottonseed oil, K 2 HPO 4 and cellulose were noted as main factors as optimized by Central Composite Design (CCD), where the culture showed highest (65.78 U/mL/min) lipase production. Findings of the research would offer potential environmental benefits along with socio-economic benefits if implemented suitably. • Response surface methodology has been reported to study Lipase production by Actinomycete • Analysis of variance (ANOVA) has been used in this study. • Highest extracellular lipase production was observed at 40°C at pH 8 of the growth medium. • Nocardiopsis alba can be used on higher scale under optimized process parameters.

Genetic Diversity and Virulence Factors of S. aureus Isolated from Food, Humans, and Animals.

Staphylococcus aureus is a commensal bacterium in humans and animals able to adapt to multiple environments. The aim of this study was to compare the genetic diversity and virulence profiles of strains of S. aureus isolated from food (29 strains), humans (43 strains), and animals (8 strains). 80 lipase-producing strains belonging to a biobank of 360 isolates, identified phenotypically as S. aureus, were selected. Confirmation of the species was made by amplifying the spA gene and 80% (64/80) of the strains were confirmed within this species. The virulence profile of each of the isolates was determined by PCR. The seA gene coding for enterotoxin A was found in 53.1% of the strains, the saK gene, which codes for Staphylokinase, was amplified in 57.8% of the strains, and, finally, the hlB gene coding for β-Hemolysin was amplified in 17.2%. The profile of antimicrobial resistance was determined by the Kirby Bauer method showing that the strains from food presented greater resistance to erythromycin (40.7%) and ciprofloxacin (18.5%) while in strains isolated from humans were to erythromycin (48.4%) and clindamycin (21.2%). Also, in strains from animals, a high resistance to erythromycin was observed (75%). The frequency of MRSA was 12.5% due to the presence of the mec gene and resistance to cefoxitin. Of the total strains, 68.7% were typed by PCR-RFLP of the coa gene using the AluI enzyme; derived from this restriction, 17 profiles were generated. Profile 4 (490 bp, 300 bp) was the most frequent, containing a higher number of strains with a higher number of virulence factors and antimicrobial resistance, which is associated with greater adaptation to different environments. In this study, a wide genetic diversity of strains of S. aureus from different foods, humans, and animals was found. This demonstrates evolution, genetic versatility, and, therefore, the adaptation of this microorganism in different environments.

Diversity of Lipase-Producing Microorganisms from Tropical Oilseeds Elaeis guineensis, Ricinus communis, and Jatropha curcas L. from Costa Rica.

Tropical oleaginous seeds are an unexplored source for the discovery of novel lipolytic microorganisms, which could be applied to the bioremediation of agro-industrial oily wastes and solve numerous environmental issues. Such wastes hold potential to be revalorized towards a variety of products through microbial bioremediation. In this study, we investigate the microbial diversity and lipase activity from bacterial and fungal isolates obtained from the oil seeds of Elaeis guineensis, Ricinus communis, and Jatropha curcas L. from Costa Rica. A total of 27 strains were confirmed as lipase-producing strains via fluorogenic and colorimetric agar plate assays. The diversity of the isolates comprises 12 fungal ascomycetes from the genera Aspergillus and Fusarium and 15 bacterial isolates classified into four genera: Serratia, Proteus, Pseudomonas, and Bacillus. Microbial isolates from E. guineensis showed the highest diversity of lipolytic microorganisms (6 genera) followed by J. curcas (4 genera) and R. communis (2 genera). Isolates showing the highest activity in agar plates were tested further by submerged fermentation and the specific lipase activity was measured with 4-nitrophenyl laurate as substrate. Accordingly, the highest specific lipase activity was demonstrated by Bacillus pumilus B5 (24.98Umg-1), Serratia marcescens B10 (17.65Umg-1), Pseudomonas mendocina B16 (8.62Umg-1), and Bacillus pumilus B1 (5.72Umg-1) in submerged fermentation. These findings indicate the presence of a specialized microbial diversity in tropical oil seeds and highlight their potential to be applied in the bioremediation of agro-industrial oily wastes.

Screening, purification and characterization of lipase from Burkholderia pyrrocinia B1213.

A lipase producing strain B1213 isolated from soil was identified as Burkholderia pyrrocinia based on 16S rRNA gene and recA sequeence analysis, making this the first report on the presence of a lipase from B. pyrrocinia. Under an aqueous two-phase purification strategy, which included (ATPE)-ion-exchange chromatography (IEC)-gel and filtration chromatography (GFC), the specific activity of the 35-kDa lipase was determined to be 875.7 U/mg protein. The optimum pH and temperature of this lipase was pH 8.0 and 50°C, respectively. The lipase retained > 85% activity in isopropanol and acetone at 30°C for 10min but the activity was reduced to 10.6% in n-hexane. Mg2+, Al3+, Mn2+, and Fe3+ enhanced lipase activity at both 1mM and 5mM concentrations. p-NPP, a long-chain acyl group 4-NP ester, appeared to be a good substrate candidate.

Growth characteristics and enzyme production optimization of lipase Producing Strain

55 samples from different regions were selected and screened by Rhodamine B flat transparent circle method to observe lipase producing effect, among which, LHY-1, identified as Serratia sp. has the characteristics of fast growth, high enzyme production and stable ability. The colony of this strain is white, the edge is smooth and tidy, the surface is moist, the cell is straight, rod-shaped, gram negative, 0.1-0.2 μm in diameter and, length 0.3-0.5 μm in length.

A novel halo-alkalo-tolerant bacterium, Marinobacter alkaliphilus ABN-IAUF-1, isolated from Persian Gulf suitable for alkaline lipase production

Alkaline lipases are one of the most important industrial enzymes that have several uses in detergents, food, pharmaceutical, cosmetics, textile, leather and biomedical industries. For screening the lipase-producing bacteria, olive agar A, B, C and Rhodamine B agar were used. The best environmental conditions and their interactions for alkaline lipase production were obtained using Taguchi statistical test. Enzyme purification was carried out by ammonium sulfate precipitation, dialysis and SDS-PAGE, respectively. The best alkaline lipase-producing strain, WS3, isolated from Persian Gulf, was named Marinobacter alkaliphilus ABN-IAUF-1 (GenBank accession number: KP403723). The maximum enzyme activity of 37.00 (μ/ml) was measured in the presence of coconut oil as the carbon source in oil broth medium after 48-h incubation at the pH 8.00 and 30 °C. The most effective factors in enzyme production were nitrogen and carbon sources with 32 and 25% of influence, respectively. Precipitation and dialysis increased the enzyme purity 1.90–2.33 times. The total enzyme activity of 925.00 ± 20.00 U in the oil broth, 96.00 ± 7.00 U after precipitation with ammonium sulfate and 60.00 ± 5.00 U after dialysis was measured. The molecular weight of alkaline lipase was measured as 55 kDa. In conclusion, this is the first report of production of alkaline lipase by a halo-alkalophilic bacterium, Marinobacter alkaliphilus ABN-IAUF-1, that was obtained from Persian Gulf, Iran. The application of enzymes produced by extremophiles such as Marinobacter alkaliphilus could be an asset in the modern environmental microbiology as well as food and industrial biotechnology.

Screening and Molecular Identification of a Bacterial Strain with Higher Lipase Productivity

The objective of this study was to screen lipase-producing strain from rich oil soil, then complete molecular identification and some culture characteristics of the isolated strain. One out of 10 strains isolated from different samples was obtained with higher lipase production, which was identified as Proteusbacillus vulgaris according to its 16S rDNA sequence and analysis phenotypic characterization together with its morphological and physiological characterization, it was nominated as Proteusbacillus vulgaris Cz-6. Some conditions of Proteusbacillus vulgaris Cz-6 for lipase production were studied in detail. The results showed that the optimal carbon source and nitrogen source in culture medium were glucose and peptone. The optimal initial pH and culture temperature were pH 5.5 and 37°C respectively. Under these conditions, after cultivated for 72 h, the measured strain’s enzyme activity grew up to 15.36 U•m L-1, which increased by 12% than the initial one before optimization.

Thermoalkalophilic lipase from an extremely halophilic bacterial strain Bacillus atrophaeus FSHM2: Purification, biochemical characterization and application

The present study was designed to isolate and identify an extremely halophilic lipase-producing bacterial strain, purify and characterize the related enzyme and evaluate its application for ethyl and methyl valerate synthesis. Among four halophilic isolates, the lipolytic ability of one isolate (identified as Bacillus atrophaeus FSHM2) was confirmed. The enzyme (designated as BaL) was purified using three sequential steps of ethanol precipitation and dialysis, Q-Sepharose XL anion-exchange chromatography and SP Sepharose cation-exchange chromatography with a final yield of 9.9% and a purification factor of 31.8. The purified BaL (Mw∼85 kDa) was most active at 70 °C and pH 9 in the presence of 4 M NaCl and retained 58.7% of its initial activity after 150 min of incubation at 80 °C. The enzyme was inhibited by Cd2+ (35.6 ± 1.7%) but activated by Ca2+ (132.4 ± 2.2%). Evaluation of BaL's stability in the presence of organic solvents showed that xylene (25%) enhanced the relative activity of the enzyme to 334.2 ± 0.6% after 1 h of incubation. The results of esterification studies using the purified BaL revealed that maximum ethyl valerate (88.5%) and methyl valerate (67.5%) synthesis occurred in the organic solvent medium (xylene) after 48 h of incubation at 50 °C.

Optimization of biological transesterification of waste cooking oil in different solvents using response surface methodology

Purpose – The purpose of this paper is to speak about the production of biodiesel from waste cooking oil which serves as an alternate fuel in the absence of conventional fuels such as diesel and petrol. Though much research work was carried out using non-edible crops such as Jatropha and Pongamia, cooking oil utilized in bulk quantity is discarded as a waste. This is reused again as it contains more of esters that when combined with an alcohol in presence of an enzyme as a catalyst yields triglycerides (biodiesel). Design/methodology/approach – The lipase producing strain Rhizopus oryzae and pure enzyme lipase is immobilized and treated with waste cooking oil for the production of FAME. Reaction parameters such as temperature, time, oil to acyl acceptor ratio and enzyme concentration were considered for purified lipase and in the case of Rhizopus oryzae, pH, olive oil concentration and rpm were considered for optimization studies. The response generated through each run were evaluated and analyzed through the central composited design of response surface methodology and thus the optimized reaction conditions were determined. Findings – A high conversion (94.01 percent) was obtained for methanol when compared to methyl acetate (91.11 percent) and ethyl acetate (90.06 percent) through lipase catalyzed reaction at oil to solvent ratio of 1:3, enzyme concentration of 10 percent at 30°C after 24 h. Similarly, for methanol a high conversion (83.76 percent) was obtained at an optimum pH of 5.5, olive oil concentration 25 g/L and 150 rpm using Rhizopus oryzae when compared to methyl acetate (81.09 percent) and ethyl acetate (80.49 percent). Originality/value – This research work implies that the acyl acceptors methyl acetate and ethyl acetate which are novel solvents for biodiesel production can also be used to obtain high yields as compared with methanol under optimized conditions.

A novel thermostable and organic solvent-tolerant lipase from Xanthomonas oryzae pv. oryzae YB103: screening, purification and characterization.

Thermostable lipases offer major biotechnological advantages over mesophilic lipases. In this study, an intracellular thermostable and organic solvent-tolerant lipase-producing strain YB103 was isolated from soil samples and identified taxonomically as Xanthomonas oryzae pv. oryzae. The lipase from X. oryzae pv. oryzae YB103 (LipXO) was purified 101.1-fold to homogeneity with a specific activity of 373.9 U/mg. The purified lipase showed excellent thermostability, exhibiting 51.1% of its residual activity after incubation for 3 days at 70 °C. The enzyme showed optimal activity at 70 °C, suggesting it is a thermostable lipase. LipXO retained 75.1-154.1% of its original activity after incubation in 20% (v/v) hydrophobic organic solvents at 70 °C for 24 h. Furthermore, LipXO displayed excellent stereoselectivity (e.e.p >99%) toward (S)-1-phenethyl alcohol in n-hexane. These unique properties of LipXO make it promising as a biocatalyst for industrial processes.

Genome sequencing and systems biology analysis of a lipase-producing bacterial strain.

Lipase-producing bacteria are naturally-occurring, industrially-relevant microorganisms that produce lipases, which can be used to synthesize biodiesel from waste oils. The efficiency of lipase expression varies between various microbial strains. Therefore, strains that can produce lipases with high efficiency must be screened, and the conditions of lipase metabolism and optimization of the production process in a given environment must be thoroughly studied. A high efficiency lipase-producing strain was isolated from the sediments of Jinsha River, identified by 16S rRNA sequence analysis as Serratia marcescens, and designated as HS-L5. A schematic diagram of the genome sequence was constructed by high-throughput genome sequencing. A series of genes related to lipid degradation were identified by functional gene annotation through sequence homology analysis. A genome-scale metabolic model of HS-ML5 was constructed using systems biology techniques. The model consisted of 1722 genes and 1567 metabolic reactions. The topological graph of the genome-scale metabolic model was compared to that of conventional metabolic pathways using a visualization software and KEGG database. The basic components and boundaries of the tributyrin degradation subnetwork were determined, and its flux balance analyzed using Matlab and COBRA Toolbox to simulate the effects of different conditions on the catalytic efficiency of lipases produced by HS-ML5. We proved that the catalytic activity of microbial lipases was closely related to the carbon metabolic pathway. As production and catalytic efficiency of lipases varied greatly with the environment, the catalytic efficiency and environmental adaptability of microbial lipases can be improved by proper control of the production conditions.

Molecular and enzymatic characterization of alkaline lipase from Bacillus amyloliquefaciens E1PA isolated from lipid-rich food waste

Bacillus amyloliquefaciens E1PA is a lipase-producing strain that was originally isolated from lipid-rich food waste, and the production of its lipase was found to be induced by vegetable oils. The E1PA lipase was successfully expressed and secreted in a heterologous Escherichia coli host and was ultimately purified. The conserved pentapeptide motif Ala-His-Ser-Met-Gly was observed at positions 108–112. The purified recombinant lipase was stable over a pH range of 4.0–11.0 at 40°C and exhibited maximal activity at pH 10. The recombinant E1PA lipase hydrolyzed a wide range of acyl esters (C4–C18). However, the highest activity (3.5unitsmg−1) was observed when the p-nitrophenyl ester of myristate (C14) was used as a substrate. Compared to the lipases produced by Bacillus spp., the E1PA lipase displayed a structural molecular mass excluding the leader sequence (19.22kDa) and a pI (9.82) that were similar to those reported for B. amyloliquefaciens lipases and lipase subfamily I.4 but that were quite distinct from those of lipase subfamily I.5 (approximately 43kDa, pI 6). These results suggested that Bacillus lipases are closely related. Although the recombinant E1PA lipase digested only certain oils, the wild-type E1PA lipase degraded a variety of oils, including blended and re-used cooking oils. The recombinant and wild-type forms of the E1PA lipase were able to digest heterogeneous lipid-rich food waste at similar levels; this result suggests that this lipase can function even when it solely consists of its structural enzyme component. The enzyme exhibited lipid hydrolysis ability as either an intracellular domain of the recombinant protein or an extracellular domain secreted by the E1PA strain. However, the recombinant lipase showed higher activity than the wild-type E1PA lipase, indicating that the recombinant protein from E. coli possessed effective lipase activity. Thus, the inducible alkaline E1PA lipase exhibited the ability to act on a broad spectrum of substrates, and the effective form produced in the heterogeneous host can be further developed for several applications, such as biodiesel production and lipase production.